Protein conjugation lies at the heart of the discovery and development of protein therapeutics. Chemical modification strategies typically employ a two step process where the first step involves site-specific modification of the protein and the second step is the installation of an entity of interest. The step involving modification of the protein can be difficult to effect site-specifically by chemical methods in view of the presence of many peptide residues of the same type. Accordingly, limited success has been achieved in such site-specific modifications, although enzymes have been targets of these transformations. For example, it has been demonstrated that group modification agents with minimal binding determinants can sometimes react site-specifically at enzyme active sites, e.g., active site serines of proteinases (Means et al., Chemical Modification of Proteins, Holden-Day, Inc., San Francisco, 1971.)
For proteins that have not evolved to do such chemistry, the challenges for site-specific labeling are far greater than for the construction of active-site directed reagents. For such proteins the challenges can be likened to the development of site-specific modifications of non-active site residues of enzymes. Thus, other than for active-sites, and allosteric sites that have evolved to bind enzyme modulators, site-specific labeling reagents (affinity labels) are lacking and novel approaches are required to fill that void. Amino acid residues usually have little to distinguish their reactivity from others in the same class, with the exception of cysteine thiols whose chemistry is quite distinct from other peptidic side chain functionality. Alternatively, strategies that employ the reaction chemistry of thiols can be useful for the selective modification of proteins, but have not been fully exploited. Current methods are limited in several respects. For example, the most commonly used thiol-specific reagent are maleimide-based, and these produce enantiomers upon reaction. Further, there are storage and stability issues with maleimide functionality as the initial adducts have a tendency to decompose over time.
For these reasons, methods for the site-specific modification and ligation of proteins would be useful for the synthesis of modified peptide, polypeptide, and protein conjugates and provide conjugates for radio-labeling, molecular imaging and protein therapeutic applications, and in methods of medical treatment that are stable and homogeneous.